This invention relates to light-detecting apparatus, and processes, using high-efficiency volume reflection holographic elements for enhancing the sensitivity of wavelength selective optical-assaying processes such as assaying by fluorimetry. One advantage that can be achieved is increasing the amount of wavelength-shifted light that can be captured from a sample being optically-evaluated. This increase is achieved while the hologram efficiently acts as a barrier to undesirable wavelengths. In some configurations of the apparatus, a hologram increases the amount of light available to illuminate the sample.
A great deal of prior art has appeared in the two areas of art which are primarily utilized in the present invention --holographic optical elements (HOEs) and fluorescent detection technology. However, HOEs have been utilized only in some types of "beam-splitting" operations. See for example, U.S. Pat. No. 3,622,220 which describes a beamsplitter using a HOE. Other beamsplitting applications appear in U.K. Patent 2,021,803A and U.S. Pat. No. 3,767,310. All of these systems pertain to beamsplitting by transmissive diffraction and utilizing different orders of diffracted light as a plurality of beams. Such procedures are different from the splitting based on wavelength discrimination as will be described below.
Holographic barrier filters (wavelength selectors) are used in some form and are described in U.S. Pat. Nos. 4,601,533; 4,669,811; 4,655,540; and 4,582,389. One application of an HOE notch-type barrier filter is to provide eye protection to military personnel against laser beams.
An enormous amount of prior art on such materials is described in U.S. Patent Office Classification Class 350-3.7 but it is believed none of the art cited therein is more pertinent to the invention claimed herein--as that cited above. In the art cited, are a large number of publications on holograms, including such publications as that in Bell System Technical Journal of November 1969 "Coupled Wave Theory for Thick Hologram Gratings" by Herwig Kogelnik (Pages 2909-2947 and the thirty-eight references cited therein).
There is also some still unpublished material, which is not statutory prior art under U.S. law, e.g. a paper given by Jon D. Masso at the SPIE-The International Society for Optical Engineering, O-E Lase '88 Conference in Los Angeles (January 1988) and entitled "Multilayer Thin Film Simulation of Volume Holograms". The information therein is useful in indicating the problems encountered in providing "conventional", multilayer, barrier filters for narrowly selected frequencies. The number of deposited layers required for a notch-type barrier filter such as illustrated in the invention to be described below would be hundreds of, perhaps thousands of, barrier layers laminated in series to achieve the desired result.
In the prior art, instruments which examine a wavelength region different from those used to illuminate a sample, such as fluorometers, will incorporate several filters to tailor the input and output wavelength regions. An exciter filter is used to isolate a particular spectral region of the light source which is to illuminate a target sample. Such a filter is not required in a detection instrument which utilizes a a single line emission from a laser as the illumination source. A second filter, or barrier filter, is then used to isolate the wavelength region of interest emitted from the sample to pass to the detection system. The ideal barrier filter would cut off all of the exciting light and pass all of the sample emitted light in the wavelength region of interest. In addition, barrier filters are used to block other kinds of "noise" light such as Rayleigh, Tyndall and Raman scattering as well as other non-specific emissions from the sample, its container and the system optical elements. These filters are typically composed of colored or tinted glass or plastic with selective absorption characteristics; gelatin dyed with organic dyes, sometimes sandwiched between two pieces of glass; or interference filters made by serial deposition of metal or dielectric films onto a glass substrate. Often a glass cover plate is cemented over a deposited stack for protection. An exciter or barrier filter of the prior art may be comprised of several of the above mentioned filters each with a different wavelength selective characteristic stacked or cemented together to achieve the desired resultant selectivity.
Instruments, which examine sample characteristics at or near epi-reflection, such as fluorescence microscopes, require some overlap of input and output light paths. To accomplish this, dichroic beamsplitters are usually employed. These beamsplitters are typically long-pass filters, usually operating at 45 degrees to the incident light, and made by deposition of metal or dielectric films onto glass substrates. Common dichroic beamsplitter designs reflect wavelengths below a given center wavelength (illumination light) with an efficiency of typically around 90%, and transmit wavelengths above the center wavelength (sample fluorescence) with around 90% efficiency.
As an example, for their IMT-2 fluorescence microscope, the Olympus Corp. (Lake Success, N.Y.) recommends a dichroic beamsplitter (sold under their trade designation DM 500) with a barrier filter (sold under Olympus designation 0 515) for use with fluorescein isothiocyanate (FITC), a well known fluorescent labelling material. In many situations, (e.g. when using an Argon-Ion laser emitting at 488 nm as the illuminating source) such a filter combination is not very efficient at capturing the emission of light from a sample to be assayed, e.g. an FITC-tagged analyte which has a peak emission around 518 nanometers.
A color filter such as the 0 515 has a relatively slow cut-on, reaching its 50% point at 515 nm and not achieving near full transmission until around 550 nm. Thus much of the usable FITC emission is lost. Attempts to avoid the problems with such conventionally recommended filters have resulted in use of interference bandpass-type filters in some instruments. These are usually comprised of a number of metal or dielectric layers and spacers forming several Fabry-Perot interferometers, or filters, stacked together. Each filter of the stack is called a "cavity". Omega Optical, Inc. of Brattleboro, Vermont manufactures multiple cavity devices, known as their "discriminating filter" series, usually with 6 to 10 cavities for use with fluorescence instrumentation. Filters with Omega Optical's trade designations of 525DF35 and 540DF65 are recommended for use as barrier filters for FITC, where the first three numbers refer to the center wavelength and the last two to the bandpass of the filter. These filters exhibit a very steep cut-on with a fairly rectangular shape to the bandpass, and good blocking of excitation light (typically to optical densities of 4-6). Although the steeper cut-on of these filters allows proportionately more of the fluorescence to be collected near the emission peak, the limited passband prevents other regions of the emission spectrum to be collected, sometimes overcoming much of the advantage gained by the steep cut-on. In addition, use of such interference filters characteristically have the problem of incurring higher transmission losses, with transmissions typically averaging around 60% of the light incident on them, and considerably less when coupled with additional out of band blocking filters.
Clearly, there remains the desire for a relatively simple filter which allows capture of more emitted light and rejection of unwanted illumination light--usually an excess of illumination light of the source wavelength.
As indicated above, an important area of art related to one aspect of the present invention is fluorescent detection technology; e.g. immunoassays wherein some analyte to be detected is labelled either directly or indirectly by any of a variety of techniques with a fluorescent material which, when stimulated by an outside light source, emits a fluorescence which is detected as a measure of the fluorescent-tagged analyte to be assayed. There is a very large amount of prior art relating to detection of fluorescence. An area of particular interest to the inventor is assays wherein only a small amount of fluorescence need be detected or is available for detection. U.S. Pat. No. 4,537,861 to Elings and Nicoli contains a discussion of prior art relative to such art as immunochemical assay.
One prior homogeneous, e.g. in situ, technique suggested for measuring such fluorescence is the technique of determining a time-rate change in fluorescent signal. This technique is neither particularly convenient nor useful when the ratio of background to the binding fluorescence to be measured is not very low. Thus, U.S. Pat. No. 4,680,275 to Wagner et al discloses only a time-delay procedure for avoiding the presence of fluorescence background in a homogeneous test method and more selectively measuring the fluorescent light emitted from the sample area.
Another technique (e.g. the invention to which U.S. Pat. No. 4,537,861 to Elings and Nicoli relates) for a homogeneous non-isotopic immunoassay is the scanning of a spatial pattern which has been created by a plurality of spaced electrodes or magnets within (or adjacent to) the biochemical composition being assayed. The scanning is carried out in such a way that one can quickly distinguish between a substantially random background fluorescent output and a substantially non-random output associated with the labelled binding reaction which one wishes to detect.
Recently-issued U.S. Pat. No. 4,683,120 shows a centrifugally-assembled "patch" of material, the geometry of which is measured as a criterion of the nature of the composition from which it is assembled.
Also U.S. Pat. Nos. 4,731,337 and 4,115,535 to Luotola and Giaever respectively, each show various procedures for immunochemical assays.
The art of stimulating fluorescent light and processing it is well known in analytical chemistry. It is well described in U.S. Pat. No. 4,675,529 to Kushida, in the above-cited Wagner Patent and others of the references cited above.
Other art which may be related to some embodiment of the invention includes the use of fluorescently-tagged beads such as magnetic beads, glass beads, polymer beads, etc. By tagged beads is meant those that are directly tagged or those to which tagged materials attach. Among the vast amount of art relating to such materials are "New Immunolatex Spheres . . . " by Molday et al in J. Cell Biology, 64, 1985; "Application of Magnetic Microsphere . . . " by Molday et al, Nature Vol 268, 1977 and "Removal of Neuroblastoma Cells . . . " by Trellaven et al, Lancet; Jan. 14, 1984.
In any event, despite a great deal of work by earlier inventors with HOEs and fluorescent-detection technology, nobody conceived of wedding the two technologies together to achieve the highly sensitive assay process and detection equipment described below. Moreover, it is believed that nobody used the HOE technologies as disclosed below to enhance both the qualitative and quantitative optical detection of light from a sample.
Of course, the above discussion of the background invention is necessarily made with hindsight knowledge of the inventions disclosed for the first time herein. It is only with that knowledge that the diverse various references and articles of manufacture cited as background herein could have been assembled and discussed as "background" for the present invention.